Repeat patient Testing-Quality control: An example of implementation in a network of commercial laboratories.

The theory and calculations underpinning Repeat Patient Testing-Quality Control (RPT-QC) have been presented in prior publications. This paper gives an example of the process used for implementing RPT-QC in a network of veterinary commercial reference laboratories and the stages associated with the transition to the sole use of RPT-QC. To employ RPT-QC in this commercial laboratory network, eight stages of implementation were identified: (1) education, (2) data collection, (3) calculations, (4) QC recording and documentation, (5) running RPT-QC in parallel with a commercially available quality control material (QCM), (6) development of a Standard Operating Procedure (SOP), (7) development of complementary aspects supporting RPT-QC, and (8) sole use of RPT-QC. Advantages of RPT-QC included cost savings for QCM and External Quality Assessment (EQA) participation and the ability to use commutable specimens with a veterinary matrix at a result level that is of clinical significance for the species. A disadvantage of RPT-QC using a single level of control was the inability to demonstrate stable performance over a range of results. Future avenues for investigation include ongoing refinement of control limits using a pooled standard deviation of the duplicates (SDdup), Sdup over time, investigation of blood samples from species other than the dog, and manipulation of specimens to produce "low abnormal" or "high abnormal" RPT-QC specimens.

Guidelines for resident training in veterinary clinical pathology. IV: Laboratory quality management-Teaching domains, competencies, and suggested learning outcomes.

BACKGROUND: The 2019 ASVCP Education Committee Forum for Discussion, presented at the annual ASVCP/ACVP meeting, identified a need to develop recommendations for teaching laboratory quality management principles in veterinary clinical pathology residency training programs. OBJECTIVES: To present a competency-based framework for teaching laboratory quality management principles in veterinary clinical pathology residency training programs, including entrustable professional activities (EPAs), domains of competence, individual competencies, and learning outcomes. METHODS: A joint subcommittee of the ASVCP Quality Assurance and Laboratory Standards (QALS) and Education Committees executed this project. A draft guideline version was reviewed by the ASVCP membership and shared with selected ACVP committees in early 2022, and a final version was voted upon by the full QALS and Education Committees in late 2022. RESULTS: Eleven domains of competence with relevant individual competencies were identified. In addition, suggested learning outcomes and resource lists were developed. Domains and individual competencies were mapped to six EPAs. CONCLUSIONS: This guideline presents a framework for teaching principles of laboratory quality management in veterinary clinical pathology residency training programs and was designed to be comprehensive yet practical. Guidance on pedagogical terms and possible routes of implementation are included. Recommendations herein aim to improve and support resident training but may require gradual implementation, as programs phase in necessary expertise and resources. Future directions include the development of learning milestones and assessments and consideration of how recommendations intersect with the American College of Veterinary Pathologists training program accreditation and certifying examination.

Repeat patient testing quality control (RPT-QC): Background and theory.

Repeat-patient testing quality control (RPT-QC) is a version of statistical quality control (SQC) in which individual patient samples, rather than commercial control materials, are used. Whereas conventional SQC assumes control material stability and repeatedly measures the same lot of control material over time, RPT-QC uses a unique patient sample for each QC event and exploits the labile nature of patient samples under prescribed storage conditions for QC purposes. Advantages of RPT-QC include commutability, lower cost, and QC at concentrations of medical interest. Challenges include sample procurement and the establishment of control limits. The objective of this review is to compare and contrast the principles and procedures of RPT-QC and conventional SQC and to provide an overview of RPT-QC control limit establishment.

Impact on result interpretation of correct and incorrect selection of veterinary glucometer canine and feline settings.

Veterinary glucometers should be correctly coded for the patient species; however, coding errors occur in clinical settings and the impact of such errors has not been characterized. We compared glucose concentrations in 127 canine and 37 feline samples using both canine and feline settings on a veterinary glucometer (AlphaTrak; Zoetis). All samples were measured first on the canine setting and then measured using the feline setting. Glucose concentration was also measured using a central laboratory biochemical analyzer (Cobas c311; Roche). Three data comparisons for each species were investigated: incorrectly coded glucometer vs. correctly coded glucometer, correctly coded glucometer vs. Cobas c311, and incorrectly coded glucometer vs. Cobas c311. For each comparison, the following analyses were conducted: Spearman rank correlation coefficient, Bland-Altman difference plot analysis, mountain plot analysis, and Deming regression. For clinical context, Clarke error grids were constructed. There was high positive correlation for all comparisons with both species. For all comparisons, mean difference was low (-0.7 to 0.5 mmol/L for canine samples, 1.0-2.0 mmol/L for feline samples). Incorrect glucometer coding resulted in proportional bias for canine samples and positive constant bias for feline samples, and individual differences could be large (-4.44 mmol/L for one dog, 6.16 mmol/L for one cat). Although the glucometer should be used per the manufacturer's recommendation, coding errors are unlikely to have severe adverse clinical consequences for most patients based on error grid analysis.

Bilateral Cubital Lymphoma and Mycobacteriosis in a Salmon-Crested Cockatoo (Cacatua moluccensis).

A 32-year-old male salmon-crested cockatoo (Cacatua moluccensis) was diagnosed by cytology with bilateral cubital lymphoma and mycobacteriosis. Polymerase chain reaction assay testing confirmed Mycobacterium genavense. This patient was subsequently humanely euthanized. Postmortem histopathology confirmed both diagnoses with findings of multicentric lymphoma, acid-fast bacilli, and severe degenerative changes in all synovial joints examined. Immunohistochemical staining for paired box protein 5 of the cubital mass was positive for a high percentage of B-cell lymphocytes, consistent with B-cell lymphoma. This unusual case of two major diseases presenting concurrently in one patient raises the question of whether the pathogenesis could have an interdependent relationship. Mycobacteriosis, severe degenerative joint changes, or both may have stimulated lymphocytes, eventually leading to lymphoma. Additional screening and monitoring for comorbidities may be advised if 1 of these diseases are diagnosed in companion avian species.

Initial investigation of molecular phenotypes of airway mast cells and cytokine profiles in equine asthma.

Equine asthma is a naturally occurring lung disease characterized by chronic, partially reversible airway obstruction, pulmonary remodeling, and lower airway inflammation. Asthma is currently divided into two major groups, mild to moderate asthma (mEA) and severe asthma (sEA), but further subtyping by phenotype (i.e., clinical presentation) and/or endotype (i.e., cellular mechanisms) may be warranted. For this study, we were interested in further investigation of cellular and inflammatory characteristics of EA, including airway mast cells. The purpose of this study was to: (1) compare mast cell protease mRNA expression between healthy and asthmatic horses, (2) analyze the cytokine profile present in BALF of currently defined equine asthma groups, and (3) use these data to evaluate potential biomarkers of defined asthma groups. We hypothesized that there would be significant differences in the cellular mast cell phenotypes (i.e., mucosal vs. connective tissue) and cytokine profiles in the BALF of asthmatic vs. healthy horses and across asthma groups. We assert these characteristics may inform additional subtypes of equine asthma. Adult horses were recruited from the institution's teaching herd and clinical caseload. Mast cell protease gene expression of the BALF cellular component and multiplex bead immunoassay for cytokine concentrations in the BALF supernatant were investigated. Airway mast cells primarily expressed tryptase, with low levels of chymase. No significant changes in protease expression were detected across groups. Horses with severe asthma had increased TNF-α, CXCL-8, and IFN-γ concentrations in BALF supernatant. Multidimensional analysis demonstrated healthy and mEA horses have overlapping characteristics, with sEA separating from the other groups. This difference was primarily due to BALF neutrophil and lymphocyte concentrations. These study results further inform understanding of EA immunopathology, and future studies designed to investigate asthma phenotypes and endotypes. Ultimately, a better understanding of these groups could help identify novel therapeutic strategies.

Analytical quality performance goals for symmetric dimethylarginine in cats.

BACKGROUND: Symmetric dimethylarginine (SDMA) reflects the glomerular filtration rate (GFR) in people, dogs, and cats. Initial assays used a liquid chromatography-mass spectroscopy (LC) technique. A veterinary immunoassay has been developed for use in commercial laboratories and point-of-care (POC) laboratory equipment. There have been no independent assessments of these assays, and analytical performance goals for SDMA testing have not been defined. OBJECTIVES: This study sought to establish analytical performance goals for SDMA in cats from (a) biological variation (BV) data and (b) expert opinion. METHODS: Analytical performance goals were determined from a prior BV study of SDMA in cats and a survey of veterinary internists who have used SDMA in practice. RESULTS: Biological variation-based performance goals included an imprecision of ±10% (immunoassay and LC), bias of ±8% (immunoassay and LC), and total error of ±24% (immunoassay and LC). Expert opinion performance goals were ±0.10 µmol/L (±2 µg/dL), or ±0.15 µmol/L (±3 µg/dL), varying with starting SDMA concentrations. CONCLUSIONS: This study recommends analytical performance goals for SDMA based on BV and expert opinion. Wide dispersion of SDMA results using currently available assays implies that clinicians risk attaching medical significance to small SDMA changes that actually reflect analytical variability.

Comparison of serum and plasma SDMA measured with point-of-care and reference laboratory analysers: implications for interpretation of SDMA in cats.

OBJECTIVES: Symmetric dimethylarginine (SDMA) reflects the glomerular filtration rate (GFR) in people, dogs and cats. Initial assays used a liquid chromatography-mass spectroscopy (LC-MS) technique. A veterinary immunoassay has been developed for use in commercial laboratories and point-of-care (POC) laboratory equipment. This study sought to: determine POC and commercial laboratory (CL) SDMA assay imprecision; determine any bias of the POC assay compared with the CL assay; calculate observed total error of the POC assay and compare with analytical performance goals; and calculate dispersion and sigma metrics (σ) for POC and CL SDMA methods. METHODS: Two separate studies were performed that assessed: (1) imprecision, determined by evaluation of pooled feline plasma or serum; and (2) bias, assessed by comparing pooled plasma and serum results, as well as paired analyses of clinical samples from a single venepuncture measured using both analysers. Results were assessed in relation to performance goals. Dispersion and σ were calculated for both analysers. RESULTS: Bias between CL and POC analysers was consistent and high numbers of clinical results were outside performance goals across both studies. Imprecision was poor for both analysers for study 1 and improved to within quality goals for the CL analyser for study 2. Dispersion was at least 40%, meaning a measured result of 14 µg/dl represents a range of possible results from 8 µg/dl to 20 µg/dl. CONCLUSIONS AND RELEVANCE: Clinicians should be careful ascribing medical significance to small changes in SDMA concentration, as these may reflect analytical and biological variability. Analyser-specific reference intervals are likely required.

Repeat patient testing-based quality control shows promise for use in veterinary biochemistry testing.

BACKGROUND: Repeat patient testing-based quality control (RPT-QC) is a form of statistical QC and an alternative to commercial quality control materials (QCM). OBJECTIVE: This study investigated the suitability of canine heparinized plasma for use in RPT-QC and assessed the predicted performance of RPT-QC for the detection of analytical error in chemistry testing. METHODS: The stability of canine plasma for RPT-QC was investigated via storage at two temperatures for three or six time points. Storage data were analyzed using repeated measures ANOVA and by comparing results for stored specimens to baseline data using predetermined criteria. To generate RPT-QC limit-setting and -validation data, leftover plasma was prospectively measured. Once control limits were established, these were challenged by measuring specimens for which the repeat aliquot had been manipulated to mimic analytical error. Finally, the predicted performance of RPT-QC and QCM-QC with four control rules was investigated using Westgard's EZ Rules 3. RESULTS: Refrigerated storage of canine plasma for 7 days allowed mild changes facilitating RPT-QC. RPT-QC limits for 12 of 17 common measurands were validated. Validated limits successfully flagged differences from manipulated specimen pairs as "error." The predicted performance of RPT-QC for analytical error detection (represented by smallest achievable allowable total error, given a probability of error detection ≥ 85% and a probability of false rejection ≤ 5%) for four common control rules is comparable to that of QCM-QC. CONCLUSIONS: This study provides evidence that RPT-QC using canine heparinized plasma refrigerated for 7 days can be used with simple control rules and low numbers of control materials, suggesting RPT-QC is applicable to both reference and in-clinic laboratory settings.

Current and emerging concepts in biological and analytical variation applied in clinical practice.

A single laboratory result actually represents a range of possible values, and a given laboratory result is impacted not just by the presence or absence of disease, but also by biological variation of the measurand in question and analytical variation of the equipment used to make the measurement. Biological variation refers to variability in measurand concentration or activity around a homeostatic set point. Knowledge of biological and analytical variation can be used to facilitate interpretation of patient clinicopathologic data and is particularly useful for interpreting serial patient data and data at or near reference limits or clinical decision thresholds. Understanding how biological and analytical variation impact laboratory results is of increasing importance, because veterinarians evaluate serial data from individual patients, interpret data from multiple testing sites, and use expert consensus guidelines that include decision thresholds for clinicopathologic data interpretation. The purpose of our report is to review current and emerging concepts in biological and analytical variation and discuss how biological and analytical variation data can be used to facilitate clinicopathologic data interpretation. Inclusion of veterinary clinical pathologists having expertise in laboratory quality management and biological variation on research teams and veterinary practice guideline development teams is recommended, to ensure that various considerations for clinicopathologic data interpretation are addressed.

Cortisol, progesterone, 17α-hydroxyprogesterone, and TSH responses in dogs injected with low-dose lipopolysaccharide.

BACKGROUND: Stress and diseases such as endotoxemia induce cortisol synthesis through a complex biosynthetic pathway involving intermediates (progesterone, and 17α-hydroxyprogesterone (17α-OHP)) and suppression of the hypothalamus-pituitary-thyroid axis. OBJECTIVE: To measure plasma concentrations of cortisol, progesterone, 17α-OHP, and thyroid stimulating hormone (TSH) in dogs experimentally injected with intravenous low-dose lipopolysaccharide (LPS). Our hypothesis was that LPS treatment would elicit a significant increase in cortisol and its precursors, and a significant decrease in TSH concentration. METHODS: Hormone measurements were performed on blood samples left over from a previous investigation (2011) on the effect of low-dose LPS on hematological measurands. Five sexually intact female dogs, none in estrous at the time of the study, were administered saline treatment two weeks prior to LPS treatment. LPS was administered intravenously at a dose of 0.1 µg/kg. Blood was collected before (baseline, time -24 hours) and 3-, 6- and 24-hours post-injection. Mixed model analysis for repeated measures was used, with both treatment and time as the repeated factors. Ranked transformation were applied when diagnostic analysis exhibited violation of normality and equal variance assumptions. Post hoc multiple comparisons were performed with Tukey's adjustment. Statistical significance was defined as p < 0.05. RESULTS: Significant differences relative to baseline values were detected following both treatments. Compared to baseline, dogs had significantly higher cortisol and 17α-OHP at 3-hours, and significantly lower TSH at 3- and 6-hours following LPS treatment. Dogs had significantly lower TSH at 6- and 24- following saline treatment. Though not statistically significant, the trend in progesterone concentrations was similar to cortisol and 17α-OHP, with an increase at 3-hours post-injection followed by a decrease close to baseline following both LPS and saline. Cortisol and 17α-OHP concentrations were higher after LPS treatment than after saline treatment at 3- and 6-hours post-injection, but differences were not statistically significant, and no significant differences between treatments were detected for any other hormone or timepoint. DISCUSSION AND CONCLUSION: Cortisol and its adrenal precursors are released in the bloodstream following a low dose of LPS, while TSH appears to decrease. Similar changes occurred following saline treatment, suggesting that even routine handling and saline injection in conditioned dogs can elicit alterations in the internal equilibrium with subsequent modification of both hypothalamus-pituitary-adrenal and thyroid axes. Changes to adrenal and thyroid hormone concentrations must be interpreted in light of clinical information. Further studies are needed to elucidate mechanisms of adrenal steroidal hormone synthesis and secretion in response to various stressful stimuli in both neutered and intact animals.

Evaluation of automated erythrocyte methodology in new world camelids using the ADVIA 2120 hematology analyzer.

BACKGROUND: Accurate erythrocyte measurements with ADVIA hematology analyzers require isovolumetric cell sphering in one reaction and hemolysis in another. However, camelid erythrocytes are resistant to sphering and osmotic lysis, and no published evaluation of ADVIA methods for camelids exists. OBJECTIVES: The objectives were to demonstrate whether camelid erythrocytes sphere in the ADVIA red blood cell/platelet (RBC/PLT) reagent and lyse in the ADVIA cyanide HGB reagent, and to determine optimal ADVIA settings for camelids. METHODS: Camelid and canine blood were diluted to 1:625 in RBC/PLT reagent and evaluated microscopically for erythrocyte sphering. A camelid sample was incubated with the hemoglobin (HGB) reagent at varying dilutions to evaluate hemolysis. The RBC, hematocrit (HCT), mean cell volume (MCV), and mean corpuscular hemoglobin concentration (MCHC) using three ADVIA species settings (equine, bovine, and caprine) were compared to their respective reference methods: Z2 Coulter impedance counter, packed cell volume, calculated MCV (PCV × 10/Coulter RBC), and calculated MCHC (HGB × 100/PCV). Reference MCV was also compared to MCV calculated using the ADVIA equine RBC count. Comparisons were assessed using Passing-Bablok regression and Bland-Altman difference plots. RESULTS: Camelid erythrocytes did not sphere in the RBC/PLT reagent, but did lyse in the HGB reagent. The ADVIA equine setting RBC count was acceptably close to the Coulter count. Hematocrit, MCV, and MCHC from all settings were significantly different from the reference methods. Mean cell volumes calculated using the equine setting RBC counts were acceptably close to the reference MCV. CONCLUSIONS: Camelid ADVIA erythrogram results should be reported as follows: RBC counts and HGB concentrations using the equine setting, spun PCVs, MCVs calculated using the PCV and equine setting RBC, and MCHCs calculated using the PCV and equine setting HGB.

Analytical quality goals-a review.

Analytical quality goals indicate how laboratory tests must perform to be clinically useful for their intended purpose. These goals have historically focused on analytical error assessment for quantitative methods and vary with measurand concentration or activity, and species. Although formalized quality goal models have been developed in human medicine, quality goals in veterinary medicine, to date, have not been formalized; use of human regulatory-based goals, consensus-based goals, or biologic variation-based goals have been reported most often. This review provides an overview of how quality goals are derived, how these may be used, and highlights challenges. Pending formal recommendations, individual veterinary laboratories should select quality goals that make the most sense clinically, logistically, and financially based on their individual needs and the needs of the clients that they serve.

Verification of the Heska Element Point-of-Care blood gas instrument for use with venous blood from alpacas and llamas, with determination of reference intervals.

BACKGROUND: The Heska Element POC ("EPOC") is a blood gas instrument intended for use with canine, feline, and equine whole blood; no verification for use with camelid specimens has been reported. OBJECTIVES: Using camelid specimens and commercial quality control materials (QCM), we investigatee EPOC analytical performance and establish EPOC camelid reference intervals (RIs). METHODS: Camelid blood (n = 124) was analyzed using the EPOC (pH, pCO2 , pO2 , HCO3 , base excess, SO2 , sodium, potassium, chloride, ionized calcium, TCO2 , anion gap, HCT, HGB, glucose, lactate, and creatinine); plasma was analyzed using a Roche Cobas c501 (sodium, potassium, chloride, TCO2 , anion gap, glucose, and creatinine). Method comparison data were assessed using Pearson's correlation, Passing-Bablok regression, and Bland-Altman plots. EPOC precision was evaluated using QCM and camelid blood. RESULTS: For all measurands except anion gap, the EPOC vs Cobas instrument correlation was r > .85. Except for pO2 and pCO2 , EPOC precision (QCM and blood) ranged from a repeatability CV <1%-6.3%. Mild constant bias for chloride, glucose, TCO2 , anion gap, and creatinine, and mild proportional bias for chloride, glucose, and anion gap were present. The total error (QCM data) for the EPOC instrument was below the ASVCP-recommended allowable total error. Alpacas had higher potassium and lactate, while llamas had higher creatinine, sodium, chloride, ionized calcium, pO2 , and SO2 . Statistical RIs based on alpaca (n = 74-96) and llama data (n = 12-17) are reported descriptively. CONCLUSIONS: The EPOC analyzer shows good performance with camelid blood. A lack of complete agreement with automated chemistry analyzers highlights the importance of interpreting patient data using instrument-specific RIs.

Repeat patient testing shows promise as a quality control method for veterinary hematology testing.

BACKGROUND: Repeat patient testing-based quality control (RPT-QC) is a potential method for veterinary laboratories (eg, that have a limited budget for quality commercial control material [QCM] or that wish to use material with a species-specific matrix). OBJECTIVES: To determine whether total error (TEa ), probability of error detection (Ped), and probability of false rejection (Pfr) similar to that achievable with QC materials can be controlled using RPT-QC METHODS: Control limits (WBC, RBC, HGB, HCT, MCV, and PLT) for the Advia 120 (n = 23) and scil Vet ABC (n = 22) were calculated using data from normal canine specimens from a routine caseload. Specimens were measured at accession and again after 24 hours. Control limits were validated using 23 additional canine specimens tested similarly. Achievable TEa, Ped, and Pfr were investigated using the Westgard EZRules3 and compared to those achievable with commercial QCM. RESULTS: Theoretical performance of RPT-QC and commercial QCM-QC are similar for 1-3s with both n = 1 and 1-3s with n = 2 for all measurands and both instruments. Achievable TEa values for RPT-QC were close to ASVCP recommendations for most measurands; exceptions were PLT (both instruments) and WBC (scil Vet ABC). CONCLUSIONS: Repeat patient testing-based quality control advantages include a species-specific matrix, low-cost, and absence of QC material deterioration over time (since a fresh specimen is used each day). A potential disadvantage is daily access to normal canine specimens. A challenge is determining control limits, which has a subjective element. Further study is needed to confirm actual RPT-QC performance and to determine if RPT-QC with abnormal patient specimens is feasible.

ASVCP guidelines: Allowable total error hematology.

The purpose of this document is to provide total allowable error (TEa ) recommendations for commonly analyzed hematology measurands for veterinary personnel. These guidelines define relevant terminology and highlight considerations specific to hematology measurands. They also provide reasons and guidelines for using TEa in instrument performance evaluation, including recommendations for when the total observed error exceeds the recommended TEa . Biological variation-based quality specifications are briefly discussed. The appendix describes the derivation of the hematology TEa recommendations and provides resources for external quality assurance/proficiency testing programs and a worksheet for implementation of the guidelines.